Glucocorticoid receptor in ayu (Plecoglossus altivelis): Genomic and non-genomic effects on monocytes/macrophages function.

The glucocorticoid receptor (GR) is an important feedback regulator of the hypothalamic-pituitary-interrenal (HPI) axis. However, there are a limited number of studies focused on host-pathogen interactions in which an association between GR and immune response has been evaluated in monocytes/macrophages (MO/MФ) after being challenged with highly pathogenic bacteria. Here, we cloned the cDNA sequence of the glucocorticoid receptor (PaGR) gene from ayu fish. The PaGR transcript was expressed in all tissues, and changes in expression were observed in immune tissues and MO/MФ after live Vibrio anguillarum infection. Subsequently, PaGR was expressed and purified to prepare anti-PaGR antibodies. We analyzed the subcellular localization of PaGR. PaGR was expressed not only in the intracellular space but also in the plasma membrane. PaGR activation decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. However, PaGR activation suppressed the phagocytosis activity of V. anguillarum-infected ayu MO/MФ via a non-genomic pathway. Interestingly, PaGR activation could enhance MO/MФ bacterial killing capability and apoptosis. Therefore, PaGR may modulate the immune response in ayu MO/MФ by genomic and non-genomic pathways.

Molecular characterization of a CC motif chemokine 19-like gene in ayu (Plecoglossus altivelis) and its role in leukocyte trafficking.

The CC motif chemokine 19 (CCL19) functions in acute inflammation by recruiting lymphocytes and other cells. However, CCL19 has only been investigated in few fish species. In this study, we characterized a CCL19-like molecule (PaCCL19l) in ayu (Plecoglossus altivelis), a teleost fish. Sequence analysis revealed that PaCCL19l was most closely related to Atlantic salmon (Salmon salar) CCL19l1, which belonged to the fish CCL19a.1 subcluster. PaCCL19l was constitutively expressed in the tested ayu tissues and peripheral blood mononuclear cells (PBMCs), with the highest transcript level in PBMCs. Upon infection with Vibrio anguillarum, the expressions of PaCCL19l in the head kidney, liver, spleen, PBMCs, and monocytes/macrophages (MO/MΦ) were dramatically up-regulated. Recombinant PaCCL19l (rPaCCL19l) exhibited a significant effect on the chemotaxis of lymphocytes and MO/MΦ in vitro and in vivo. Meanwhile, rPaCCL19l exerted a high chemotaxic activity for lipopolysaccharide (LPS)-stimulated MO/MΦ (M1-type), but not for cyclic adenosine monophosphate (cAMP)-stimulated MO/MΦ (M2-type). When ayu MO/MΦ was treated with rPaCCL19l along with Vibrio anguillarum infection, the mRNA expression of proinflammatory cytokines (IL-1ß, TNFα, IL-6, IL-12b, and IFN-γ) was up-regulated, while that of anti-inflammatory cytokines (IL-10, TGFß, and IL-22) was down-regulated. Ayu MO/MΦ treated with anti-PaCCL19l IgG gave the opposite result. These results implicated that PaCCL19l is involved in the selective chemotaxis of ayu immune cells and promotes the host at a pro-inflammatory state.

Molecular characterization of a tissue factor gene from ayu: A pro-inflammatory mediator via regulating monocytes/macrophages.

Tissue factor (TF) plays an important role in the host's immune system as the principal initiator of coagulation. However, the precise function of TF in teleosts remains unclear. We determined the cDNA sequence of TF from ayu Plecoglossus altivelis (PaTF). The PaTF transcript was expressed in all tested tissues, and changes in expression were observed in tissues and monocytes/macrophages (MO/MФ) upon infection with Vibrio anguillarum. PaTF was prokaryotically expressed and purified to prepare anti-PaTF antibodies. Western blot analysis revealed that native PaTF was glycosylated in thrombocytes, but not in ayu MO/MФ. Microparticles could transfer PaTF to thrombocytes. PaTF neutralization or knockdown led to anti-inflammatory status in ayu MO/MФ upon V. anguillarum infection. PaTF neutralization reduced the apoptosis of ayu MO/MФ and improve survival rate in V. anguillarum-infected ayu. Our results indicate that PaTF plays a role in ayu immune response against bacterial infection as a pro-inflammatory mediator.

A Novel Lipopolysaccharide Recognition Mechanism Mediated by Internalization in Teleost Macrophages.

Macrophages in teleosts are less sensitive to lipopolysaccharide (LPS) compared to mammals. The functional equivalent of the mammalian LPS surface receptor in teleost macrophages for the pro-inflammatory response is either non-existent or replaced by negative regulation. LPS signaling in teleost macrophages remains unclear. Here, we found a scavenger receptor class B 2a (PaSRB2a) that played a crucial role in LPS signaling in teleost macrophages. The internalization of LPS and subsequent pro-inflammatory responses in macrophages were mediated by PaSRB2a, which is a novel isoform of the mammalian SRB2 gene. LPS internalization by PaSRB2a is dependent on its C-terminal intracellular domain. Following LPS internalization, it interacts with the ayu intracellular receptors nucleotide-binding oligomerization domain protein 1 (PaNOD1) and PaNOD2. Moreover, LPS pre-stimulation with sub-threshold concentrations reduced the effect of secondary LPS treatment on pro-inflammatory responses that were mediated by PaSRB2a. The pro-inflammatory responses in LPS-treated ayu were down-regulated upon PaSRB2a knockdown by lentivirus siRNA delivery. In grass carp and spotted green pufferfish, SRB2a also mediated LPS internalization and pro-inflammatory responses. Our work identifies a novel LPS signaling pathway in teleosts that differs from those in mammals, and contributes to our understanding of the evolution of pathogen recognition in vertebrates.